This application is a U.S. national phase of PCT/BE98/00183, filed Nov. 25, 1998, which claims priority of Belgium Application Serial No. 9700949, filed Nov. 26, 1997.
The present invention relates to a process for in vitro inhibition of the production of cytokines by cells, in particular animal or human cells, and of the secretion thereof.
The object of the present invention is to provide a process for in vitro inhibition of the production of cytokines which does not jeopardise said cells. Advantageously, this process will allow inhibition of the production and secretion of substances promoting the appearance of immunoallergic phenomena in these cells.
This object is achieved according to the invention by a process as described above comprising application onto said cells of at least one azo derivative of the formula 
in which A represents a carboxyl group or a group 
B represents a carboxyl group or a group 
R1, R2, R3 and R4 are identical or different and each mutually independently represent a hydrogen or halogen atom, an optionally substituted aliphatic or aromatic hydrocarbon residue or a hydroxy group, R1 and R2 possibly being joined together to form a heterocyclic nucleus with the nitrogen atom adjacent thereto, and R3 and R4 possibly being joined together to form a heterocyclic nucleus with the nitrogen atom adjacent thereto, X1 and X2 are identical or different and each mutually independently represent an oxygen atom or a group NR5, in which R5 is a hydrogen or halogen atom, an optionally substituted aliphatic or aromatic hydrocarbon residue or a nitro group, and in which, when two groups NR5 are simultaneously present, each R5 may be identical to or different from the other, together with the salts, esters and isomers thereof.
Various of these azo derivatives are compounds which are known in particular for the antiviral activity thereof, especially against viruses of the retrovirus group, in particular the AIDS virus (c.f. WO-A-9116054 and WO-A-9107876, together with U.S. Pat. No. 5,585,367).
More particularly in relation to 1,1xe2x80x2-azobisdimethylformamide, which is also known as diamide, many researchers have studied the activation phenomenon of the intracellular transcription factor NF-kappaB and have demonstrated that diamide blocks the function of certain enzymes in the activation cascade for this factor.
Other authors have demonstrated that at certain concentrations diamide induces apoptosis in certain cell lines.
Still other authors have demonstrated that diamide has an effect on certain membrane receptors.
However, none of these studies reveals any inhibitory action of diamide on the production of cytokines by the cells under observation. It may even be noted that C. Mendez et al. in xe2x80x9cOxidants augment endotoxin-induced activation of alveolar macrophagesxe2x80x9d, SHOGK, volume 6, no. 3, pp. 157-163, 1996, observe an insignificant increase in the production of tumour necrosis factor at a dose of 1 mmol, i.e. the reverse effect to that repeatedly observed on various cytokines according to the present invention.
It is moreover known that the azo derivatives according to the invention exhibit extremely low intrinsic toxicity towards the human body or healthy cells which are treated.
According to one embodiment of the invention, the process comprises in vitro inhibition of the production and secretion of interleukins by cells. Interleukins which may in particular be mentioned are interleukins IL-2, IL-4, IL-5, IL-6, IL-8, IL-10, IL-12 and IL-15, with inhibition of IL-2 and IL-5 most particularly being intended.
According to another embodiment of the invention, the process comprises in vitro inhibition of the production and secretion of interferon-xcex3 (IFN-xcex3) by cells. According to still another embodiment, the process comprises inhibition of the production and secretion of tumour necrosis factor xcex1 (TNF-xcex1).
According to an advantageous embodiment of the invention, in the above-stated general formula, R1 to R5 each represent an aliphatic or aromatic hydrocarbon residue comprising 1 to 6 carbon atoms. The heterocycles optionally formed in the general formula may contain, apart from one nitrogen atom, at least one other heteroatom, for example oxygen. The heterocycle is, for example, pentagonal to octagonal, preferably being hexagonal. The azo derivative according to the invention may be selected from among the group comprising azobisformamidine derivatives, such as 1,1xe2x80x2-azobisform-amidine, 1,1-azobisnitroformamidine, 2,2xe2x80x2-azobismethyl-formamidine, 1,1xe2x80x2-azobisfluoroformamidine, 1-monochloro-azobisformamidine, azobis [chloroformamidine], azobisformamide derivatives, such as 1,1xe2x80x2-azobisformamide and dimethylazobisformamide, 1,1xe2x80x2-(azodicarbonyl)-dipiperidine, 1,1xe2x80x2-(azodicarbonyl)dimorpholine, azodihydroxamic acid and the salts thereof, azodicarboxylic acid and the salts thereof.
The process advantageously comprises application of the azo derivative onto the cells at a dose which does not induce apoptosis thereof. A micromolar concentration of approx. 0.4 to approx. 200, preferably of 2 to 20, advantageously of 2 to 10, may be considered.
It this regard, 1,1xe2x80x2-azobisdimethylformamide, also known as diamide, may under certain circumstances exhibit the disadvantage of bringing about cellular apoptosis at certain excessively high concentrations, while providing a relatively short half-life of only a few minutes.
According to one embodiment of the invention, at least one of the stated azo derivatives is applied onto cells isolated from macroorganisms or cells of microorganisms, for example originating from cell cultures. The treated cells may also be those from an organ or multicellular tissue taken from the body of a human or an animal, such as for example the cells from a sample of blood or lymph.
According to one embodiment of the invention, at least one of the stated azo derivatives is applied, for example, onto cytokine producing cells isolated from human blood. These cells may be extracted in an overall manner, in which case overall effects will be observed on the various types of lymphocytes, on cells having antigens and on macrophages in co-cultures. A more advanced stage of the investigation may consist in measuring the production of cytokines by highly purified cell lines, for example CD4 lymphocytes.
The cell lines concerned may also be treated by administering azo derivatives to living organisms, including human beings, and measuring the various groups of cytokines in the biological fluids taken from the body of a human or an animal before, during and/or after treatment, and by measuring the production capacity for the lymphokines under consideration after extraction of the cells present in these fluids.
It is also possible to consider treating grafts or cellular tissues, prior to the transplantation thereof into the body of a human or an animal, with an azo derivative according to the invention.
It is also possible to provide the use of pharmaceutical preparations based on azo derivatives according to the invention to treat patients against graft rejection.
Cytokines are produced in the body by different cell reservoirs.
It is known, for example, that lymphocytes specialise specifically in the production of cytokines and that, depending upon the type of cytokine produced, a distinction is drawn between Th1 and Th2 CD4 lymphocytes.
Cytokines are also produced not only by CD8 lymphocytes (IL-4 and IL-5), but also by mast cells and eosinophils and, during uterine implantation, by the ectodermal cells of the trophoblast.
Cytokines are produced by these various cell reservoirs during the appearance of various inflammatory and allergic reactions in general and during the phenomenon of graft rejection. The following is a non-exhaustive list of pathological conditions which may inter alia be mentioned: asthma, atopic dermatitis, allergic seasonal rhinitis, psoriasis, pemphigus, thyroiditis, myasthenia gravis, rheumatoid arthritis, IgA nephropathy, scleroderma, lupus erythematosus, insulin-dependent diabetes, disseminated sclerosis, inflammatory diseases of the digestive tract, Crohn""s disease, hypereosinophilia, eosinophilic syndrome, and any condition due to IL-5 mediated cellular apoptosis.
The active substance according to the invention may be applied onto cells alone or mixed with other substances according to the invention, or also mixed with other substances having a different effect on the cells. The active substance(s) may also be applied as such or in the form of a composition comprising at least one of the azo derivatives according to the above-stated formula and an appropriate carrier or vehicle.
Certain compounds according to the invention have at least one asymmetrical C atom and consequently all isomers, including diastereomers and rotational isomers or enantiomers, are also provided by the invention. The invention includes the D and L isomers in pure or mixed form, including racemic mixtures.
Certain acid-type compounds, for example carboxylic acids, may form salts, for example with metals, or with acceptable amines, or esters with compatible alcohols.
The present invention also relates to the use of at least one of the derivatives of the above-stated formula, together with the salts and isomers thereof, for the production of pharmaceutical preparations for use in the treatment or prevention of human or animal conditions arising from pathological cellular production of said cytokines. It is in particular possible to provide the production of pharmaceutical preparations for the treatment and/or prevention of autoimmune and/or inflammatory conditions involving T lymphocytes, allergic inflammatory diseases or also the rejection of allografted and/or xenografted organs and the graft-versus-host reaction after a cellular allograft. Said use will thus advantageously be provided for producing pharmaceutical preparations intended for use in the treatment or prevention of conditions such as those stated above.
The pharmaceutical preparation produced according to the invention may be administered by any appropriate route, inter alia orally, sublingually, rectally or vaginally, by injection or perfusion, locally, transcutaneously or via the mucous membranes. The pharmaceutical preparation contains a therapeutically effective quantity of the above-stated azo derivative(s). Dosage will vary from individual to individual depending upon their own immunological characteristics, which are in part genetically determined. The pharmaceutical preparation may be presented as any appropriate dosage form, for example as capsules, pills, tablets, coated tablets, powders, injectable forms, creams, ointments, known transdermal administration systems, inhalable preparations.
The pharmaceutical formulations and compositions may contain conventional pharmaceutically acceptable excipients, optionally together with conventional pharmaceutical additives. These excipients and additives in particular comprise compatible inert fillers, binders, disintegration agents, buffers, preservatives, antioxidants, lubricants, flavouring agents, thickeners, colours, emulsifiers etc.
Since the primary application is therapeutic, the present invention also provides azo derivatives of the above-stated general formula in which at least one of the groups A and B represents a carboxyl group, together with the pharmaceutically acceptable salts, esters and isomers thereof, for the application thereof as therapeutically active substances. Derivatives of this type which may in particular be considered are azodicarboxylic acids of the following formula 
and the salts, esters and isomers thereof.
Similarly, the present invention also relates to azo derivatives of the above-stated general formula, in which the group A represents the group 
one of the residues R1 and R2 representing a hydroxy group and the other a hydrogen atom, and in which the group B may simultaneously represent the group 
one of the residues R3 and R4 possibly representing a hydroxy group and the other a hydrogen atom, together with the pharmaceutically acceptable salts, esters and isomers thereof, for the application thereof as therapeutically active substances. We have in mind in particular the azodihydroxamic acid of the following formula 
and the salts and isomers thereof.
The invention finally furthermore relates to 1,1xe2x80x2-(azodicarbonyl)dimorpholine of the formula 
together with the salts, esters and isomers thereof, for the application thereof as therapeutically active substances.
The invention also relates to products containing at least one of the azo derivatives of the above-stated formula and the isomers thereof and at least one cytokine as a combination product for use simultaneously, separately or in a staggered manner in the therapy or prevention of human or animal conditions arising from pathological cellular production of at least one cytokine differing from said cytokine, at least one of which is present in the combination product, or in the therapy or prevention of human conditions, a side effect of which is pathological cellular production of at least one cytokine differing from said cytokine, at least one of which is present in the combination product.
Further embodiments and developments of the invention are stated in the attached claims.